30% Effort. Genomic and proteomic approaches to understand oral cancer Although risk factors for HNSCC, such as alcohol and tobacco consumption, are well recognized, the molecular mechanisms responsible for this malignancy are still not fully understood. We have used a number of novel approaches to investigate gene and protein expression profiles in HNSCC. We have shown that laser capture microdissection (LCM) can be used to procure specific cell populations from heterogenous tumor samples, and that LCM-procured material can be used effectively to extract RNA, DNA, and proteins. We have teamed up with other research institutions to conduct gene and protein expression analysis of HNSCC by combining LCM, gene arrays, and proteomic platforms. These efforts have already provided a wealth of information about the distinctive pattern of gene and protein expression in HNSCC. Gene and protein expression analysis: In prior gene array analysis efforts, we have identified numerous genes that were differentially expressed between normal oral mucosa and cancer, and many distinct genes when comparing cancers associated with betel quid chewing and tobacco use. This body of information enabled us to identify a large number of molecules whose contributions to HNSCC progression are now under investigation in our laboratory and in the extramural community. Examples of molecules that were evaluated during this reporting period include sphingosine kinase, COX-2 and prostaglandin receptors, EPS8, and FOXM1. These studies also revealed that the expression of VEGF-C, a potent lymphangiogenic growth factor, is a shared feature of the most metastatic HNSCC lesions, which provided the rationale for current efforts investigating whether interfering with VEGF-C can halt the metastatic spread of HNSCC. Carbon nanotubes: immunosensensors for cancer biomarkers and drug delivery systems for cell-surface receptor-guided cancer therapy: The genomic and proteomic analysis of HNSCC may now allow the development of novel biomarkers of diagnostic and prognostic value. We continued teaming up with J. Rusling at UCONN to develop nanotube-based systems for the detection of premalignant lesions and micrometastasis in sentinel lymph nodes. We succeeded in using single-wall carbon nanotube (SWNT) forest platforms for immunosensing biomarkers in serum and tissue lysates, and for the targeted killing of HNSCC cells, taking advantage of the ease of functionalization of SWNTs. As most HNSCC overexpress EGFR, we attached cisplatin, a first line chemotherapeutic agent, and EGF (for targeting) to SWNTs. EGF-SWNTs were internalized by HNSCC cells depending on the surface overexpression of EGFR. EGF-SWNT-cisplatin bioconjugates caused the rapid death of HNSCC cells, but sparing control cells showing low levels of EGFR. These studies provided the first evidence of targeted killing of cancer cells by guiding SWNTs loaded with an anticancer drug to cancerous cells based on cell surface receptors. We followed this study by performing the first single-molecule imaging of platinum-based drug attached to single-wall carbon nanotubes, a collaborative achievement that has recently received the broad attention of the emerging nanomedicine community. 40% Effort.Dysregulated signaling networks in HNSCC: novel mechanism-based approaches for HSNCC prevention and treatment There is an urgent need for new treatment options for HNSCC patients, considering that their overall 5-year survival is relatively low (50%) and has not improved much over the past 3 decades. The emerging information on the nature of the deregulated molecular mechanisms responsible for HNSCC progression has provided the possibility of exploring new mechanisms-based therapeutic approaches for HNSCC. For example, we have observed that persistent activation of the serine-threonine kinases mTOR is frequent event in HNSCC, and that inhibition of mTOR by the use of rapamycin causes the rapid decrease in the level of pS6 (a downstream target of mTOR) and the apoptotic death of HNSCC tumor xenografts, thereby causing tumor regression. These efforts have identified the Akt-mTOR pathway as a potential therapeutic target for HNSCC. Inhibition of mTOR by rapamycin prevents lymphangiogenesis and locoregional lymph node metastasis in a new HNSCC orthotopic model. HNSCCs often metastasize to locoregional lymph nodes, and lymph node involvement represents the most important prognostic factor of poor clinical outcome. Of interest, in collaboration with Bhuvanesh Singh, MSKCC, we observed that the activation of mTOR represents a widespread event in human clinical specimens of HNSCC invading locoregional lymph nodes. Furthermore, both primary and metastatic experimental HNSCC lesions exhibited elevated mTOR activity. To begin exploring the contribution of mTOR to HNSCC metastasis we developed an orthotopic model consisting in the implantation of fluorescently labeled HNSCC cells into the tongues of immunecompromised mice. These orthotopic tumors spontaneously metastasize to the cervical lymph nodes, where the presence of HNSCC cells can be revealed by histological evaluation. Together with Roberto Weigert, we visualized the tumoral cells invading the tongue and within the lymphatic vessels using intravital two-photon microscopy. The ability to monitor and quantitate lymph node invasion in this model system enabled us to explore whether the blockade of mTOR could impact on HNSCC metastasis. We found that inhibition of mTOR with rapamycin diminished lymphangiogenesis in the primary tumors and prevented the dissemination of HNSCC cancer cells to the cervical lymph nodes, thereby prolonging animal survival. These findings may provide a rationale for the future clinical evaluation of mTOR inhibitors, including rapamycin and its analogs, as part of a molecular-targeted metastasis preventive strategy for the treatment of HNSCC patients. 30% Effort. Animal models for oral malignancies A major limitation in the area of HNSCC research is the limited availability of animal models to test the validity of current genetic paradigms of tumorigenesis, and to explore the effectiveness of treatment modalities or chemopreventive approaches. Novel genetically-defined and chemically induced oral-specific animal models to study SCC: We have recently made significant contributions to the development of genetically engineered mouse models (GEMM) for HNSCC. We have continued with these studies, including the recent analysis of the interplay between the TGF-&#946;and Akt-mTOR pathway in collaboration with Ashok Kulkarni. In this line of research, we have now focused our efforts in this area in the development of oral specific systems enabling the activation/inactivation of genes in the epithelial stem cell compartment. Current studies are aimed at recapitulating HNSCC progression, thus providing a suitable system to investigate targeted approaches to halt tumor development. In addition to the use of Cre-recombinase and tetracycline-inducible systems to delete relevant tumor suppressor gene products (i.e., p16, Pten, p53, TGF-&#946;receptors) and to express oncogenic molecules (i.e., Ras, EGFR), we are now making a new effort in two different directions. We are attempting to develop a high throughput animal model by the use of lentiviruses that are only active in the epithelial stem cell compartment. This approach may position us to address the biological relevance of newly identified genetic alterations that are expected to result from the oncogenomic studies in our intra-and extra-mural community. For this line of research, we are also exploring the use of new AAV serotypes based on our ongoing collaborations with Jay Chiorini.